Summary of Work: Cytochromes P450 metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) in the heart. In humans, rats, and mice, the predominant cardiac enzymes responsible for EET production are CYP2J2, CYP2J3, and Cyp2j6, respectively. We have shown that micromolar concentrations of 11,12-EET significantly improve cardiac contractility following prolonged, global ischemia in isolated-perfused rat hearts. In contrast, other CYP2J products (e.g. 14,15-EET and 19-HETE) are inactive in this system, demonstrating the specificity of the biological effect for 11,12-EET. In isolated-perfused rabbit papillary muscle preparations, 11,12-EET causes hyperpolarization of the resting membrane potential and significant shortening of the cardiac action potential; during ischemia, 11,12-EET decreases extracellular K+ accumulation and conduction velocity consistent with a preconditioning-like effect. Some of these electrophysiologic effects are explained by the observation that 11,12- EET directly affects cardiac L-type Ca++ channels incorporated into artificial lipid bilayers. In order to study the effects of increased EETs on cardiac function and on the physiologic response of the heart to ischemia-reperfusion in vivo, we have begun to construct transgenic mice that overexpress human CYP2J2. We have successfully subcloned the CYP2J2 cDNA into the pBS2-_MHC-hGH vector which contains the _-myosin heavy chain promotor to drive cardiac-specific expression of the transgene. The linearized DNA will be microinjected into pronuclei of single cell fertilized mouse embryos, the eggs implanted into pseudopregnant female mice, and the offspring screened for the presence of the CYP2J2 transgene by PCR/Southern blot analysis. Founder animals will be screened for heart-specific expression of CYP2J2 by Northern and Western blotting. In order to examine the effects of reduced EETs on cardiac function in vivo, we have begun to prepare mice in which the Cyp2j6 gene has been disrupted. We have cloned and sequenced the mouse Cyp2j6 cDNA and have cloned the corresponding mouse Cyp2j6 gene. Currently, we are sequencing the gene to characterize intron/exon boundaries and are in the process of preparing the knock-out construct.